The Anti-Atopic Dermatitis Effects of Mentha arvensis Essential Oil Are Involved in the Inhibition of the NLRP3 Inflammasome in DNCB-Challenged Atopic Dermatitis BALB/c Mice

The NLRP3 inflammasome is upregulated by various agents, such as nuclear factor-kappa B (NF-κB), lipopolysaccharide (LPS), and adenosine triphosphate (ATP). The NLRP3 inflammasome facilitations the maturation of interleukin (IL)-1β, a proinflammatory cytokine that is critically involved in the pathogenesis of atopic dermatitis (AD). Although the NLRP3 inflammasome clearly exacerbates AD symptoms such as erythema and pruritus, drugs for AD patients targeting the NLRP3 inflammasome are still lacking. Based on the previous findings that Mentha arvensis essential oil (MAEO) possesses strong anti-inflammatory and anti-AD properties through its inhibition of the ERK/NF-κB signaling pathway, we postulated that MAEO might be capable of modulating the NLRP3 inflammasome in AD. The aim of this research was to investigate whether MAEO affects the inhibition of NLRP3 inflammasome activation in murine bone marrow-derived macrophages (BMDMs) stimulated with LPS + ATP in vitro and in a murine model displaying AD-like symptoms induced by 2,4-dinitrochlorobenzene (DNCB) in vivo. We found that MAEO inhibited the expression of NLRP3 and caspase-1, leading to the suppression of NLRP3 inflammasome activation and IL-1β production in BMDMs stimulated with LPS + ATP. In addition, MAEO exhibited efficacy in ameliorating AD symptoms in a murine model induced by DNCB, as indicated by the reduction in dermatitis score, ear thickness, transepidermal water loss (TEWL), epidermal thickness, and immunoglobulin E (IgE) levels. Furthermore, MAEO attenuated the recruitment of NLRP3-expressing macrophages and NLRP3 inflammasome activation in murine dorsal skin lesions induced by DNCB. Overall, we provide evidence for the anti-AD effects of MAEO via inhibition of NLRP3 inflammasome activation.


Introduction
The skin is constantly exposed to pathogens and environmental risks, including pollutants, particulate matter (PM), and heavy metals, and is prone to skin disorders [1]. Atopic dermatitis (AD) is a common skin disorder that is characterized by symptoms such as erythema, redness, swelling, dryness, and persistent itching. The prevalence of AD has increased in recent years due to urbanization and industrialization, with many experiencing

The Effects of MAEO on NLRP3 Inflammasome Formation in BMDMs
We investigated the effects of MAEO on the expression of NLRP3 and the formation of inflammasomes in LPS-primed BMDMs by pretreating cells with different concentrations of MAEO (12.5-100 µg/mL) before stimulation with LPS (500 ng/mL, 4 h) and exposure to ATP (50 mM) for an additional 30 min. LPS + ATP-primed cells showed increased NLRP3 expression, whereas 100 µg/mL MAEO pretreatment significantly suppressed NLRP3 expression ( Figure 1A). NLRP3 inflammasome formation converts pro-caspase-1 into its active form, leading to the cleavage of pro-IL-1β into mature active IL-1β [27]. Therefore, we investigated the expression of caspase-1 and IL-1β to confirm that MAEO can regulate NLRP3 inflammasome formation. As expected, pretreatment with MAEO dose-dependently reduced the LPS + ATP-induced increase in active caspase-1 ( Figure 1B). Similarly, LPS + ATP-stimulated cells expressed active IL-1β, which disappeared in both the pro-and active forms above 50 µg/mL MAEO ( Figure 1C). We noted that MAEO considerably decreased the production of the proinflammatory cytokines IL-1β and IL-18, which are induced by NLRP3 inflammasomes (Figure 1D,E). Additionally, similar to our prior research, MAEO also reduced the level of IL-6 in the culture medium ( Figure 1F). These findings suggest that MAEO may have anti-inflammatory properties by controlling NLRP3 expression and regulating inflammasome formation. MAEO was pretreated 30 min before the induction of inflammation, and LPS (500 ng/mL) was used to stimulate inflammation for 4 h followed by ATP (50 mM) addition. Relative protein expression of (A) NLRP3, (B) caspase-1, and (C) IL-1β was quantified. The levels of (D) IL-1β, (E) IL-18, and (F) IL-6 were determined in culture medium. One-way ANOVA was used for statistical analysis, and significance was established as # p < 0.05, ## p < 0.01, ### p < 0.001 versus the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the LPS + ATP-stimulated group. MAEO was pretreated 30 min before the induction of inflammation, and LPS (500 ng/mL) was used to stimulate inflammation for 4 h followed by ATP (50 mM) addition. Relative protein expression of (A) NLRP3, (B) caspase-1, and (C) IL-1β was quantified. The levels of (D) IL-1β, (E) IL-18, and (F) IL-6 were determined in culture medium. One-way ANOVA was used for statistical analysis, and significance was established as # p < 0.05, ## p < 0.01, ### p < 0.001 versus the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the LPS + ATP-stimulated group.

The Effects of MAEO on DNCB-Induced AD-like Clinical Symptoms in BALB/c Mice
Based on the in vitro studies, we investigated the effects of MAEO on the expression of NLRP3 in AD skin lesions using a DNCB-induced AD-like murine model. The SCORAD index and ear thickness were used to evaluate the DNCB-induced AD-like clinical symptoms at 4-day intervals. Treatment with 1% MAEO topically resulted in significant recovery of AD symptoms, which was evident from Day 12 and continued until the final day, as shown in Figure 2A. The SCORAD index, which quantifies clinical symptoms, showed a significant increase in the control group that was treated with the vehicle in contrast to the normal group. As expected, MAEO effectively decreased the dermatitis score, ear edema, and TEWL ( Figure 2B-D). We also observed that MAEO decreased the level of IgE in plasma, an important biomarker in determining AD ( Figure 2E). In addition, the levels of IL-18 and IL-6, which play central roles in inflammation, were significantly decreased by 1% MAEO (Figure 2F,G). Furthermore, we observed that MAEO was successful in decreasing the level of TSLP, a proinflammatory cytokine that leads to AD ( Figure 2H).
Furthermore, we performed histological examinations, including H&E and TB staining, to assess the effects of MAEO on epidermal thickness and mast cell infiltration in the dorsal skin tissue of mice with AD-like symptoms induced by DNCB. The control group had considerably higher levels of epidermal thickness and infiltrated mast cells than the normal group. However, MAEO significantly decreased the DNCB-induced increase in epidermal thickness and infiltrated mast cells ( Figure 2I,J). Notably, MAEO effectively decreased the expression of IL-1β infiltration in the dorsal skin tissue of mice with DNCB-induced AD-like symptoms ( Figure 2K). These results support and corroborate previous research indicating a significant amelioration of DNCB-induced atopic skin inflammation with 1% MAEO treatment, and a potential link to the modulation of NLRP3 inflammasome activity.

The Effects of MAEO on NLRP3 Inflammasome Formation in Macrophages of DNCB-Induced AD-like Murine Dorsal Skin
Macrophages, the main cell type mediating the NLRP3 inflammasome response, can massively migrate to skin with AD lesions. Therefore, we identified that MAEO has the potential to obstruct inflammasome formation and NLRP3 expression in macrophages found in AD lesions ( Figure 3A,B). The DNCB-induced AD model demonstrated a notable increase in the count of cells positive for NLRP3 and F4/80, whereas administration of 1% MAEO significantly attenuated NLRP3-expressing macrophages ( Figure 3C). In addition, we observed an increase in double-stained cells positive for NLRP3 and ASC in the dorsal skin of DNCB-induced AD-like mice. As expected, the group treated with 1% MAEO exhibited a statistically significant reduction in the number of cells positive for both NLRP3 and ASC ( Figure 3D). These findings indicate that MAEO can effectively regulate both the infiltration of NLRP3-overexpressing macrophages and formation of the NLRP3 inflammasome in DNCB-induced AD lesions.    MAEO significantly attenuated NLRP3-expressing macrophages ( Figure 3C). In addition, we observed an increase in double-stained cells positive for NLRP3 and ASC in the dorsal skin of DNCB-induced AD-like mice. As expected, the group treated with 1% MAEO exhibited a statistically significant reduction in the number of cells positive for both NLRP3 and ASC ( Figure 3D). These findings indicate that MAEO can effectively regulate both the infiltration of NLRP3-overexpressing macrophages and formation of the NLRP3 inflammasome in DNCB-induced AD lesions.

Discussion
We demonstrated the anti-inflammatory properties of MAEO, specifically related to the NLRP3 inflammasome, in both LPS + ATP-induced BMDMs and an AD murine model induced by DNCB. AD is a persistent inflammatory condition of the skin that is influenced by several cofactors, including impaired skin barrier function, immune system modifications, and a complex genetic background [28]. The aforementioned factors contribute to the disruption of the epithelial structure, leading to increased epidermal thickness, swelling, redness, and formation of lichenized plaques in AD [29,30]. Inflammatory skin lesions in AD are known to be characterized by the accumulation of macrophages, and many studies on AD have reported that macrophage accumulation is a crucial factor in its pathogenesis [31][32][33]. Excessive and prolonged inflammation activates an inflammatory complex called the NLRP3 inflammasome in macrophages in skin lesions.

Discussion
We demonstrated the anti-inflammatory properties of MAEO, specifically related to the NLRP3 inflammasome, in both LPS + ATP-induced BMDMs and an AD murine model induced by DNCB. AD is a persistent inflammatory condition of the skin that is influenced by several cofactors, including impaired skin barrier function, immune system modifications, and a complex genetic background [28]. The aforementioned factors contribute to the disruption of the epithelial structure, leading to increased epidermal thickness, swelling, redness, and formation of lichenized plaques in AD [29,30]. Inflammatory skin lesions in AD are known to be characterized by the accumulation of macrophages, and many studies on AD have reported that macrophage accumulation is a crucial factor in its pathogenesis [31][32][33]. Excessive and prolonged inflammation activates an inflammatory complex called the NLRP3 inflammasome in macrophages in skin lesions.
The inflammatory response heavily relies on the NLRP3 inflammasome, which includes the adapter molecule ASC, responsible for procaspase-1 recruitment. This inflammasome is primarily activated in proinflammatory macrophages, causing the release of proinflammatory cytokines, such as IL-1β and IL-18 [34]. Therefore, IL-1β and IL-18 might be significant mediators of the AD phenotype through NLRP3 inflammasome activation [35]. IL-1β is a strong stimulator of immune and inflammatory responses that cause immune cell recruitment and activation at sites of inflammation or infection [36]. Numerous studies have reported that IL-1β contributes to skin inflammation in AD and induces IL-6 production both in vitro and in vivo [37][38][39][40]. The improper regulation of IL-6 triggers a cytokine storm associated with various autoimmune disorders, such as AD and asthma [41]. Additionally, IL-18, which belongs to the IL-1 family together with IL-1β, has the ability to regulate immune responses [42]. In fact, IL-6 and IL-18 have been suggested as potential etiologies of AD, as their blood levels have been shown to increase with AD severity [43][44][45][46]. Therefore, regulating the NLRP3 inflammasome and its associated cytokines, including IL-1β, IL-18, and IL-6, is important in preventing inflammatory skin diseases. Moreover, some previous studies have suggested that the NLRP3 inflammasome ultimately triggers pyroptosis, leading to various skin diseases, including AD and acne [14,47]. Mitochondrial cleavage inhibitor 1 (mdivi-1), an NLRP3 inflammasome inhibitor, has been proposed as a therapeutic agent for AD due to its ability to inhibit NLRP3 inflammasome activation and pyroptosis in keratinocytes under AD-like inflammation [48,49]. mdivi-1 was reported to decrease expression of NLRP3 and ASC, cleavage of caspase-1, and mature IL-1β and IL-18 in keratinocytes under AD-like inflammation [49]. Therefore, targeting the NLRP3 inflammasome through inhibitors such as mdivi-1 may be a promising therapeutic approach for AD. Notably, BMDMs can induce inflammation (M1-polarized macrophages) through the release of proinflammatory cytokines in response to various stimuli, such as LPS and endogenous stress. Although the role of LPS in atopic dermatitis is still unknown, there is evidence that LPS-activated NF-κB can determine the severity of AD [50,51]. Additionally, excessive Th1 due to Th1/Th2 imbalance can activate M1-polarized macrophages and worsen AD [52]. Furthermore, ATP acts on P2X7 receptors, a subtype of purine receptor, to induce K + efflux and affect NLRP3 inflammasome formation [53]. In particular, BMDMs are utilized as a suitable model because K + efflux is a general requirement to activate the NLRP3 inflammasome [54][55][56][57]. The findings of this investigation demonstrated that MAEO inhibited the upregulation of NLRP3 and cleavage of caspase caused by LPS + ATP. In addition, MAEO lowered mature IL-1β and production of the proinflammatory cytokines IL-1β and IL-18. Furthermore, MAEO reduced the production of IL-6, which is essential in innate immunity together with the NLRP3 inflammasome. Based on our observations, we hypothesized that MAEO may have the potential to modulate AD by inhibiting development of the NLRP3 inflammasome in BMDMs.
Expanding on the previous findings, we investigated the potential therapeutic effects of MAEO against AD by assessing its impact on macrophage infiltration and NLRP3 inflammasome activation in vivo. The topical application of MAEO was administered to DNCB-induced AD-like lesions in BALB/c mice, which were repeatedly treated with DNCB on their dorsal skin and ear. As expected, excellent relief effects of 1% MAEO were visually observed and quantified as an index. The DNCB-induced increases in dermatitis score, ear thickness, and TEWL were significantly decreased by 1% MAEO compared to the control group. In addition, 1% MAEO reduced the levels of IgE, IL-18, IL-6, and TSLP in plasma. The main immunological changes in AD are an increase in both IgE and hypersecretion of cytokines [58]. Concomitantly, the level of TSLP in the blood may also increase due to an allergic reaction [59]. Many studies have reported significantly increased levels of IgE, IL-18, IL-6, and TSLP in the plasma of patients or animals with AD [44,45]. In addition, topical MAEO administration had a positive effect on epidermal thickness reduction and mast cell infiltration in the dorsal skin. Additionally, we confirmed that 1% MAEO decreased IL-1β expression in dorsal skin-induced AD. Thus, MAEO was expected to reduce NLRP3 inflammasome activation in AD-like dorsal lesion skin. Furthermore, we found increased accumulation of NLRP3-expressing macrophages and formation of the NLRP3 inflammasome in DNCB-induced AD-like dorsal skin lesions. In particular, confirmation of co-localization of macrophages (F4/80) with NLRP3 and NLRP3 with ASC suggests a link between activation of the NLRP3 inflammasome in macrophages and the severity of AD. MAEO reduced the accumulation of NLRP3-expressing macrophages and development of the NLRP3 inflammasome in dorsal skin lesions. Moreover, 1% MAEO significantly downregulated the expression of the NLRP3 inflammasome in comparison to the control group. In addition to our in vitro results in BMDMs, in vivo results allow us to understand better the mechanisms by which NLRP3 inflammasome expression in macrophages affects AD severity. Thus, MAEO has the potential to suppress both inflammation and NLRP3 inflammasome activation, ultimately leading to its anti-AD effects (Figure 4). substances and has already been used; thus, MAEO has the advantage of being applicable to humans [60,61]. Therefore, MAEO can be considered as an alternative or complementary therapy to current treatments for AD, such as topical corticosteroids and immunosuppressive agents, to enhance their effectiveness and reduce the side effects. Taken together, these results indicate that MAEO holds considerable promise as a therapeutic intervention for remedying inflammatory skin disorders, with a particular focus on atopic dermatitis, through its capacity to suppress inflammasome activity.  Figure 4. Proposed mechanism for the action of MAEO on the NLRP3 inflammasome under inflammatory conditions induced by LPS + ATP and AD-like conditions induced by DNCB. The mechanism of action of MAEO for MAPK/NF-κB inhibition was reported in our previous study [23].
In conclusion, we investigated the effects of MAEO on the activities of the NLRP3 inflammasome in LPS + ATP-induced inflammatory stimulation and DNCB-induced AD-like murine models. As we reported, MAEO, containing menthol and menthone as its primary constituents, possesses potent anti-inflammatory properties and may be a promising candidate for treating AD. Additionally, MAEO could be formulated as a cream or lotion and applied to affected areas to reduce inflammation and alleviate symptoms. Moreover, we found that MAEO effectively ameliorated AD-like symptoms by modulating expression of the NLRP3 inflammasome. Although clinical trials on NLRP3 inflammasome inhibition by MAEO are still lacking, we identified the anti-inflammatory molecular mechanism of MAEO and its ameliorating effect on atopic symptoms through two studies. Modulating the expression of NLRP3 inflammasome by MAEO suggests that inhibiting its activity could be a potential therapeutic strategy for treating inflammatory skin disorders such as psoriasis and eczema. In addition, MAEO is generally considered to contain safe substances and has already been used; thus, MAEO has the advantage of being applicable to humans [60,61]. Therefore, MAEO can be considered as an alternative or complementary therapy to current treatments for AD, such as topical corticosteroids and immunosuppressive agents, to enhance their effectiveness and reduce the side effects. Taken together, these results indicate that MAEO holds considerable promise as a therapeutic intervention for remedying inflammatory skin disorders, with a particular focus on atopic dermatitis, through its capacity to suppress inflammasome activity.

Mentha arvensis Essential Oil
As previously described by Kim et al. [23], MAEO was prepared. Our previous research demonstrated that MAEO exhibited anti-inflammatory properties without cytotoxicity at 100 µg/mL in both RAW 264.7 macrophages and HaCaT keratinocytes. In addition, 1% MAEO had anti-atopic effects in the DNCB-induced AD murine model. Therefore, to ensure consistency with previous experiments [23], we used the same concentration for all investigations in this study. For in vitro treatment, MAEO was dissolved in dimethyl sulfoxide to a final concentration of 0-100 µg/mL, resulting in a transparent solution with a unique spicy scent. For in vivo experiments, MAEO was dissolved in olive oil at a concentration of 1% (v/v) and vortexed, resulting in a sticky, translucent yellow solution with a spicy scent. The solvent used affected the color and viscosity of the resolution, while the distinctive spicy scent was consistent.

Gas Chromatography-Mass Spectrometry
The Kim et al. [23] method was used to determine the various compounds present in MAEO. MAEO was analyzed using a Varian CP3800 gas chromatograph and a Varian 1200 L mass detector (Varian, Inc., Palo Alto, CA, USA) fitted with a polydimethylsiloxanemodified VF-5MS capillary column (30 m × 0.25 mm × 0.25 µm). The sample was heated from 50 • C to 250 • C at a 5 • C/min rate in the oven. The ionization detector and injector temperature were adjusted to 200 • C and 250 • C, respectively. Helium was employed as the carrier gas, with a constant flow rate of 1 mL/min. A split ratio of 10:1 was used to inject 2 µL of the sample, and the mass spectrum was obtained using a 70 eV electron ionization energy system that scanned the range of 50-500 m/z. The compound was identified by comparing its linear retention index (LRI) obtained from gas chromatography with reference spectra from the National Institute of Standards and Technology (NIST, 3.0) and available literature data [62]. Chemical standards, including menthol, menthone, and piperitone were obtained from Sigma Chemical Co. (St. Louis, MO, USA). To quantify the major compounds in MAEO, a standard solution containing the main compound was injected in a volume of 1.0 µL at an appropriate concentration. The major constituents were determined in triplicate, and their concentrations were determined by plotting calibration curves using their peak areas.

Animals
The animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines of the Laboratory Animal Research Center at Kangwon National University, Korea (KW-200122-2). Six-week-old female BALB/c mice (14-15 g) were procured from the Orient Bio Experimental Animal Breeding Center (Seongnam, Republic of Korea) and housed in cages with 5 animals each under standard conditions, including a 12 h light-dark cycle, a constant humidity of 45-65%, and unrestricted access to food and water. A total of 30 mice were utilized for the study, with 10 mice allocated to each group.

Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA kits from R&D Systems (Minneapolis, MN, USA) and Invitrogen (Carlsbad, CA, USA) were used to determine the levels of IL-1β, IgE, IL-18, IL-6, and TSLP in the culture medium or plasma, following the manufacturer's instructions. The collected cell culture medium and plasma were used as samples after centrifugation at 1500 rpm and 10,000 rpm for 5 min, respectively. The optical density at 450 nm was measured using a SpectraMax 190 microplate reader (Molecular Devices, San Jose, CA, USA).

DNCB-Induced AD Mice
A previous study [23] provided an experimental plan to investigate the impact of MAEO on DNCB-induced AD-like mice. In brief, the dorsal skin of the mice was shaved using shaving cream and a clipper, and the mice were divided into three groups of ten mice each: a group that received no treatment (normal), a group that was sensitized with DNCB (control), and a group that was treated with 1% MAEO. To cause AD, DNCB, a compound that causes allergic dermatitis in mice, was diluted to 1% in a mixture of acetone and olive oil (3:1). The dorsal skin and ears of mice were sensitized with 200 µL and 20 µL of 1% DNCB twice a week, resulting in the appearance of AD symptoms, including erythema, edema, and papulation [65]. Seven days after shaving, the dorsal skin and ears were treated daily with either 1% MAEO or olive oil. To maintain AD symptoms, 0.4% DNCB was applied every other day for 2 weeks. The progress of AD symptoms was evaluated every 4 days by measuring the dermatitis score, ear thickness, and transepidermal water loss (TEWL). The assessment of DNCB-induced AD lesions was conducted by applying the SCORing Atopic Dermatitis (SCORAD) index, which scored the severity of erythema, edema, excoriation, and lichenification of the skin on a scale from 0 (no lesion) to 3 (severe) [66]. Ear thickness was measured with a digital micrometer (Mitutoyo Co., Tokyo, Japan) and the TEWL was evaluated using a GPSKIN barrier research solution-II (Gpower Inc., Hanam, Republic of Korea).

Histological Observation
As mentioned in the experimental schedule, the dorsal skin tissue of each mouse was collected on the final day. The skin tissues were preserved in 10% formalin and subsequently embedded in paraffin before being cut into sections [67]. Each section was stained with hematoxylin and eosin (H&E) and toluidine blue (TB), and light microscopy (Olympus, Tokyo, Japan) was used to observe and analyze the histological images. Epidermal thickness was evaluated at a magnification of 200× by examining the H&E-stained sections [68]. The number of mast cells, evaluated by TB staining, was counted in three randomly chosen sections [69].

Statistical Analysis
The measurements were performed in triplicate and expressed as the means ± standard error of the mean (S.E.M.). The data analysis was conducted using GraphPad Prism Version 8.0 (GraphPad, La Jolla, CA, USA). Two-way analysis of variance (ANOVA) of one-way ANOVA, followed by a Student-Newman-Keuls test, was used for multiple comparisons, and statistical significance was considered at p < 0.05.  Abbreviations AD, atopic dermatitis; Dexa, dexamethasone; DNCB, dinitrochlorobenzene; IL, interleukin; NF-κB, nuclear factor-kappa B; LPS, lipopolysaccharide; ATP, adenosine triphosphate; MAEO, Mentha arvensis essential oil; BMDMs, bone marrow-derived macrophages; NLRs, nucleotide-binding oligomerization domain-like receptors; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain.